The overall objective of this research is to understand the structural basis for the production of a functional cytochrome P450 (P450) molecule, which must 1) be targeted to an retained in the endoplasmic reticulum (ER), 2) have orientation relative to the membrane compatible with interactions of P450 with P450 reductase and other ER membrane proteins and 3) have binding sites for its substrates, heme, O2, and P450 reductase. Our previous work has demonstrated that the hydrophobic N- terminal region of P450 is responsible for its insertion into the ER and is sufficient for its retention there. Objectives in this proposal related to cellular localization are to define the minimal N-terminal sequence required for retention in the ER by deletion analysis of N- terminal sequence fused to reporter genes and expressed in COS-1 cells, to test whether retention is due to a specific signal in this region or is a default condition by analyzing chimeric proteins of P450 and a plasma membrane protein, epidermal growth factor receptor, and to identify the signal, if present, by mutagenesis, and characterize its receptor in the ER by cross-linking studies. To examine determinants of membrane orientation, the effect of mutations in the region connecting the transmembrane and cytoplasmic catalytic domains, a proline/glycine rich sequence, and internal hydrophobic regions, which may interact with membrane lipids, on cellular localization, enzymatic activity and interaction with P450 reductase will be analyzed. Substrates of rabbit P450 2Ci and P450 2C2 include lauric acid, arachidonic acid and other fatty acids, and a chimeric 2C2/2C1 has a novel progesterone C21- hydroxylase activity. To identify amino acids that may be in one of the six substrates recognition sites (SRS), based on alignments of the sequence with the 3-dimensional structure of bacterial P50cam, the effect of systematic mutations in SRS-1, SRS-5 and SR-6 regions on metabolism of these substrates will be examined. Mutations will be screened for activity in COS-1 cells, and those with diminished activity will be expressed in E. coli or yeast for more detailed spectral and kinetic analysis. P450s are critical proteins in human medicine. They are responsible for the detoxification of xenobiotic agents, the biogenesis of endogenous compounds, and the activation of carcinogens and mutagens. P450s are involved in important drug interactions, polymorphic rates of drug metabolism and congenital disease, such as adrenal hyperplasia. These studies on the normal cell biology should provide insight on P450's role in pathological states.